Currently, when drugs are conventionally administered to a patient, they circulate throughout the entire body of the patient. As a result, extremely high dosages are required to reach therapeutic levels in the desired organ. This non-targeted delivery of high dosages of drugs results in systemic toxicity and severe side-effects.
Targeted delivery of therapeutic or diagnostic agents to specific organs or tissues is much safer and more effective than delivery of a drug to an entire individual, as is the case by conventional administration techniques. The ability to specifically deliver a composition (e.g., a drug or gene) to a specific organ or tissue in vivo allows much smaller amounts of the drug to be administered thereby reducing associated side effects.
Conventional means to achieve this sort of “targeted” or organ-specific delivery includes the use of implants (e.g., Elisseeff (1999) Proc. Natl. Acad. Sci. USA 96:3104-3107), stents or catheters (see, e.g., Murphy (1992) Circulation 86:1596-1604), or vascular isolation of an organ (e.g., liver, see, e.g., Vahrmeijer (1998) Semin. Surg. Oncol. 14:262-268). However, these techniques are invasive, traumatic and can cause extensive inflammatory responses and fibrocellular proliferation (see, e.g., van der Giessen (1996) Circulation 94:1690-1697).
A more sophisticated strategy is the targeted delivery of compounds to organ- or tissue-specific molecules exposed on the luminal surface of the vasculature. Previous attempts at tissue-specific delivery depended on sites within the tissue that were inaccessible to the compounds due to the natural barrier of the vasculature. Hence the importance of identifying accessible, tissue-specific molecules on the luminal surface of the vasculature. For example, vasculature-targeted chemotherapy, i.e., the destruction of tumor blood vessels with cytotoxic agents, makes use of biochemical differences between angiogenic and resting blood vessels (see, e.g., Ruoslahti (1999) Adv. Cancer Res. 76:1-20). This approach may minimize or eliminate some of the problems associated with conventional solid-tumor targeting, such as poor tissue penetration and drug resistance. Eliminating tumor blood supply using anti-angiogenic agents can have dramatic anti-tumor effects. Targeting chemotherapeutic agents to the tumor vasculature kills tumor blood vessels in addition to having the usual anti-tumor activities of the drug. This approach can result in increased efficacy and reduced toxicity of anti-tumor agents.
However, the versatility and scope of any biochemical targeting strategy is dependent on the in vivo or in situ identification of organ- or tissue-specific molecules as expressed on the luminal surface of the vasculature. One strategy is to identify organ-specific molecular differences in vivo is by screening peptide libraries expressed on the surface of bacteriophage (see, e.g., Rajotte (1998) J. Clin. Invest. 102:430-437). This method, though, will miss many potential tissue-specific molecules because it is dependent on the ability of fusion proteins to bind to cell surface molecules with sufficient affinity to isolate such molecules.
Another strategy is to selectively radioiodinate lumen-expressed polypeptides in situ. (see, e.g., Schnitzer (1990) Eur. J. Cell Biol. 52:241-251). However, this method is limited because it only labels polypeptides containing tyrosine residues and does not facilitate isolating the labeled molecule.
Another approach coats lumen-exposed cells with cationized silica particles followed by polyanion crosslinkers, in situ. (see, e.g., Schnitzer, et al., U.S. Pat. Nos. 5,281,700; 5,776,770; 5,914,127). The tissue is then homogenized and cell membranes bound to the silica are isolated by density gradients. This method, though, results in a significant fraction of non-lumen-expressed molecules contaminating the isolated fraction. In the Schnitzer-silica particle technique, once the cells are homogenized, all intracellular molecules can bind to the silica-polyanion complex. When whole membranes are isolated with this technique, molecules not exposed to the luminal surface are also isolated.
Another approach used in situ was to label isolated lungs by perfusing the pulmonary artery with the non-cleavable cell membrane impermeant biotinylation reagent sulfosuccinimidyl 6-biotin-amido hexanoate, which labels amine groups of polypeptides (De La Fuente (1997) Amer. J. of Physiol. 272:L461-L470). Tissue homogenates were incubated with strepavidin-agarose beads. To elute biotinylated polypeptides from the strepavidin harsh conditions were used because the affinity between biotin and avidin is about 10−15 M−1. This resulted in significant contamination with non-specifically binding polypeptides and other non-lumen exposed molecules in the eluate. This method is also flawed in that significant amounts of naturally biotinylated proteins not normally exposed to the lumen in vivo are also isolated.
Because of the increased demand for use of more sophisticated drug delivery techniques, such as the biochemical strategy of targeted delivery of drugs and genes only to organ- or tissue-specific molecules, different ways of identifying and isolating such molecules are needed. The present invention addresses these and other needs.